Antimicrobial Efficacy of Leaf Extracts of Piper Nigrum Against Escherichia Coli, Staphylococcus Aureus and Candida Albicans
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Kigigha LT et al. Antimicrobial activity of P. nigrum leaves 32 JOURNAL OF BASIC Original Research Article PHARMACOLOGY AND TOXICOLOGY Antimicrobial efficacy of leaf extracts of Piper nigrum against Escherichia coli, Staphylococcus aureus and Candida albicans Lovet T. Kigigha, Chidinma Grace Kalunta* Department of Biological Sciences, Faculty of Science, Niger Delta University, Wilberforce Island, Bayelsa state, Nigeria. Abstract This study investigated the antimicrobial activity of Piper nigrum leaves extracts (hot water, cold water and pepper soup) on Escherichia coli, Staphylococcus aureus and Candida albicans. The fresh leaves of climbing pepper were bought at Swali Market, Yenagoa, Bayelsa State of Nigeria. Hot and cold-water extracts and pepper soup made with the leaves were used for the sensitivity test. Filter paper disc method was used for the determination of zone of inhibition. Ampiclox (1%) was used as the positive control (AMP). The mean zone of inhibition for S. aureus, E. coli and C. albicans were 12.52 mm, 12.61 mm and 11.23 mm respectively (cold water leave viz: CWL), 13.16 mm, 13.57 mm and 11.49 mm respectively (hot water leave viz: HWL), 13.46 mm, 13.60 mm and 13.31 mm respectively (pepper soup extract leave viz: PEPL) and 12.07 mm, 12.31 mm and 12.03 mm respectively (1% concentration of the ampiclox used as positive control viz: AMP). Analysis of variance showed that there was a significant variation (p < 0.05) among the various isolates for each of the different extracts apart from PEPL. The phytochemical screening revealed the presence of tannins, flavonoids, cardiac glycosides and alkaloids in the leaves of P. nigrum. Keywords: Antimicrobials, Microorganisms, Plant extracts, Leaves of Piper nigrum of a variety of diseases caused by microorganism especially 1. Introduction in developing countries [3, 7, 8]. Authors have variously reported that over 80% of world population depend on Due to the challenges of drug resistance, there have been traditional medicine practitioners for healthcare services [3, an upsurge in search for effective alternative to sythenthic 9-12]. In developing countries, individuals, who patronize drugs. According to Pandey et al. [1], the development of herbal medicine practitioners, are mostly in the rural areas effective antibacterial agents are needed due to the [6]. emergence of drug-resistant organisms because of the Medicinal plants are plants whose roots, leaves, seeds, irrational and overuse of antibiotics, failure to complete a bark, or any other constituent possess therapeutic course of treatment, genetic versatility of microbes and characteristics [3, 4, 6, 10]. Again, plants are source of food horizontal transfer of resistant genes among bacterial including carbohydrates, proteins, minerals, vitamins. As species. In addition, the emerging and re-emerging diseases such, they are essential for the sustenance of life. have also contributed to effective search of new antibiotics. Plants are also used as spices in preparation of delicacies. Over the past few decades, the natural antioxidants of both According to Roy et al. [13], spices are natural food nutritive and medicinal plants have been of significant additives which are known to contribute immensely to the interest to the pharmaceutical and food industries due to taste of food and are also known to possess tremendous their roles in combating myriads of oxidative damages medicinal properties. Several types of spices are used in incurred by living cells and food products from free developing countries like Nigeria. Some notable spices used radicals’ activities [2]. Recently, there have been increasing include clove, nutmeg, garlic, ginger, various varieties of interests in the discovery of new antimicrobials, because of pepper such as Piper nigrum. the increasing risk in the rate of infection with antibiotic- Piper nigrum, which belongs to Piperaceae family [5] is resistant microorganisms, and also due to the side and the popularly known as black pepper and has culinary and residual effects of antibiotics. Plants have emerged as medicial uses [13]. Medicinal properties of Piper nigrum credible alternative [3,4] or a promising effect in have been comprehensively reviewed and documenetd by therapeutics [5] due to their natural properties that can be Ahmad et al. [14], Damanhouri et al. [15]. This could be due easily absorbed. to several organic chemicals including essential oil, The use of plant to treat human diseases can be traced to alkaloids, flavonoids, phenols, lignans, acids, amides and several centuries [6]. Till date, a large number of global other aromatic compounds it contains [13]. population still depend on traditional medicine for treatment *Corresponding author; E-mail: [email protected] J Basic Pharmacol Toxicol. 2017;1(2):32-36 33 Piper nigrum is often referred to as ‘‘The King of cooker) and allowed to boil until the final quantity of water Spices’’ [13, 15, 16] and have gained worldwide popularity obtained was 20 mL. It was filtered using a muslin cloth on in the spice industry [17]. Due to the bioactive and metabolites the plant contains, it has inordinate potential for the discovery of novel biologically active compounds [17]. Like other plants, Piper nigrum has antimicrobial potentials [1, 13- 24]. But, information about the comparison of the different extracts with pepper soup extract is scanty in literature. Hence, this study aimed at assessing the antimicrobial efficacy of leaf extracts of piper nigrum against enteric, superficial, etiologic microorganisms such as Escherichia coli, Staphylococcus aureus and Candida albicans. 2. Materials and methods 2.1. Source of plant samples and identification The fresh leaves of climbing pepper were bought at Swali Market, Yenagoa, Bayelsa State of Nigeria at the early hours Figure 1. Pictorial nature of the Pepper soup extract of Piper nigrum of the day (6:30 am - 7:00 am) in the month of June 2015. The samples were botanically identified using the a funnel and further filtered using Whatman No.1 filter morphological characteristics presented by Nyananyo [25]. paper. The various extracts were also stored in the refrigerator at 5 °C prior to use. Figure 1 presents the 2.2. Preparation of plant materials for extraction pictorial nature of the pepper soup extract. The collected fresh plant materials were transported to 2.4. Experimental procedures the laboratory of the Department of Biological Sciences, Faculty of Sciences, Niger Delta University within 1 hr. The The various extracts i.e. hot water, cold water and pepper plant leaves were picked from the stems, cleaned and rinsed soup were used for the antimicrobial activity testing viz. in deionized water. The leaves were allowed to air dry. cold water leaves (CWL) and hot water leaves (HWL). 2.3. Extraction processes 2.5. Source and preparation of organisms One hundred grams (100 g) of the leaves was weighed The fresh isolates of Staphylococcus aureus, Escherichia and rinsed three times in deionized water and ground into a coli and Candida albicans used in this study were obtained fine powder in a sterilized porcelain mortar. Two different from the stock culture from the Medical Microbiology and solvents were used i.e. cold and hot water. Ten grams (10 g) Parasitology Department, College of Health Sciences, Niger of fine grounded Piper nigrum leaf powder was measured Delta University, Nigeria. The purity of the bacteria was out using electronic balance (Series JT 302N) and then checked by sub-culturing and the resultant growth was transferred into a conical flask containing 50 mL of sterile subjected to biochemical test using the scheme of Benson hot water at 96 ± 4 °C (which reduced to room temperature [26] and Cheesbrough [27]. Germ tube test was carried out after about 2 hr). It was corked with a cotton wool and foil, for the Candida albicans. shaken gently and allowed to stand in room temperature for 24 hr. The content was then transferred to a funnel bearing 2.6. Preparation of antibiotic disc and antibiotics medium a sterile muslin cloth and further filtered using a Whatman No. 1 filter paper. This process was repeated for the cold Antibiotic discs were prepared using a 6 mm diameter water extract at room temperature of 29 ± 3 °C. All the paper-perforating machine on Whatman No. 1 filter paper. extracts were stored in the refrigerator at 5 °C prior to use. Then, 0.1 mL of extract was dropped on a sterilized 7 mm In the pepper soup extract, the initial quantity of water in Whatman No. 1 filter paper placed in sterilized petri dishes cooking the pepper soup was 300 mL. Thirty grams of and was allowed to be absorbed in room temperature. After smoked fish (Heterobranchus bidorsalis which belong to drying, they were safely packed in a petri-dish, wrapped in the Clariidae family) was thoroughly washed and put inside an aluminum foil and stored in the refrigerator. a pot containing 300 mL of cold water. This was followed The positive control antibiotic used was 500 mg by 5 g of pepper, salt to taste, a cube of seasoning (Maggi), Ampiclox (broad-spectrum antibiotic containing ampicillin a small ball of onion and a teaspoon of grounded crayfish 250 mg and cloxacillin 250 mg) and the media used was the (Macrobrachium felicinum which belong to the multipurpose medium (nutrient agar) for Staphylococcus Palaemonidae family) and then 10 g of fine grounded seed aureus, MacConkey agar for Eschericia coli and chocolate powder of piper nigrum. It was put on burning flame (gas dextrose agar for Candida albicans. The contents of 500 mg Kigigha LT et al. Antimicrobial activity of P. nigrum leaves 34 ampiclox capsule were dissolved in 500 mL of water giving nitrogen compounds (monoterpenes, diterpenes, triterpenes, out 1 mg/ml solution [28]. tetraterpenes, sesquiterpenes, saponins, flavonoids, steroids and coumarins) [2, 42]. 2.7. Antimicrobial screening of the extract The occurrence of these phytochemicals alkaloids, flavonoids, tannins and cardiac glycosides suggested the Filter paper disc method previously described by Benson usefulness of the leaves against microorganisms.